Chicken antibodies to a recombinant fragment of the equine immunoglobulin epsilon heavy-chain recognising native horse IgE.
An equine immunoglobulin E (IgE) heavy-chain cDNA fragment (CH3-CH4, nucleotides 1132 to 1592) was cloned, expressed in Escherichia coli as a fusion protein with a [His]6-tag and purified over a Ni-NTA column. The recombinant protein was used to immunise hens. Testing of the raised egg yolk immunoglobulin G (IgG) in Western-blot and ELISA revealed high titres against the recombinant equine IgE fragment (reqIgEf). The reqIgEf-specific IgG was successfully affinity-purified on an unconventional affinity matrix: the [His]6-tagged recombinant IgE fragment was bound to Ni-NTA agarose and used to adsorb specific immunoglobulins. In Western-blot of ammonium sulphate precipitated horse serum and bronchoalveolar lavage fluid, separated by SDS-PAGE under denaturing-reducing conditions, the raised antibodies reacted with a protein of approximately 80 kDa. A reaction of the reqIgEf-specific IgG was seen with a 190-200 kDa band when the same horse serum or bronchoalveolar fluid (BALF) was separated under non-reducing conditions. These reactions could be inhibited by preincubation of the immune IgG with reqIgEf, while preincubation with horse IgG did not inhibit the reaction. Antibody-affinity chromatography of horse serum with the reqIgEf-specific chicken IgG resulted in an enrichment of the 80 kDa protein in denaturing Western-blot. Determination of the amino acid composition of this protein and comparison with the equine IgE heavy- chain sequence strongly indicates that the 80 kDa band corresponds to the heavy chain of the horse IgE. The reqIgEf-specific chicken IgG was further characterised in an ELISA for the detection of allergen-specific horse IgE. It was demonstrated that heating IgE positive horse sera at 54 degrees C for 10 min drastically diminished the recognition by the reqIgEf-specific chicken IgG. The reaction is inhibitable by preincubation with reqIgEf in a concentration dependent manner. In addition, preincubation with horse IgG, a nonrelevant [His]6-tagged protein or 2% equine colostrum had no influence on the reqIgEf-specific chicken IgG binding characteristic. This antibody recognising horse IgE will be useful for further studies on the pathogenesis of equine allergic diseases.
Marti
E
E
Klinik für Nutztiere und Pferde, University of Berne, Switzerland. marti@itz.unibe.ch
Peveri
P
P
Griot-Wenk
M
M
Muntwyler
J
J
Crameri
R
R
Schaller
J
J
Gerber
H
H
Lazary
S
S
D000596:Amino Acids / Q000032:analysis; D000707:Anaphylaxis / Q000276:immunology / Q000662:veterinary; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D002645:Chickens; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003126:Colostrum / Q000276:immunology; D004926:Escherichia coli; D006041:Goats; D006736:Horses; D006358:Hot Temperature; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D007139:Immunoglobulin epsilon-Chains / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007202:Indicators and Reagents; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000596:Amino Acids; D000888:Antibodies, Anti-Idiotypic; D007139:Immunoglobulin epsilon-Chains; D007202:Indicators and Reagents; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E